Enzyme intended for the fragmentation of N-acetylheparosan, production of preparations containing this enzyme and fragmentation process using this enzyme

ABSTRACT

The invention relates to an enzyme capable of fragmenting a high molecular mass N-acetylheparosan. This enzyme was obtained with the strain Escherichia coli (K5), strain SEBR 3282.

This is a divisional application of application Ser. No. 08/020,396,filed Feb. 22, 1993 now U.S. Pat. No. 5,482,844.

The present invention relates to an enzyme intended for thefragmentation of N-acetylheparosan, to the production of preparationscontaining this enzyme and also to fragmentation processes using thisenzyme.

Some bacteria of the species Escherichia coli are known to produce acapsular polysaccharide, usually referred to as K5, which is a family ofpolymers consisting of repeatedβ-D-glucuronyl-(1→4)-N-acetyl-α-D-glucosaminyl-(1.fwdarw.4) units (W. F.Vann et al, Eur. J. Biochem, 1981, 116, 359-364), of structure (a):##STR1##

This polysaccharide will be referred to here as "N-acetylheparosan".This product possesses a molecular mass of between 10⁵ and 2×10⁶ Da and,in respect of the "uronic acid" units, a very regular structure composedsolely of D-glucuronic acid (W. F. Vann et al, Eur. J. Biochem, 1981,116, 359-364, and Patent Application EP-A-0,333,243).

N-Acetylheparosan is, most particularly, useful as a starting materialfor the pharmaceutical industry but, for this use, it has too high amolecular mass.

It is known that N-acetylheparosan (polysaccharide K5) may be fragmentedby a phage lyase originating from a phage specific for the strainEscherichia coli (K5), but this fragmentation is very extensive andleads to a substantial disappearance of the fragments of molecular mass5000 Da in favour of much smaller chains (D. Gupta et al, FEMSMicrobiology Letters, 1983, 16, 13-17). This fragmentation is used inPatent Application EP-A-0,333,243 for the preparation of fragmentscontaining at most 10 saccharide units.

It is also known that oligosaccharides possessing at the non-reducingend a glucuronic unit having double bond between carbons 4 and 5 may beobtained by enzymatic depolymerisation of polysaccharides, using as anenzyme the enzyme obtained from Bacillus circulans, and in particularfrom Bacillus circulans strain NCIB 12482. This enzyme acts on theglucose-glucuronic acid bonds (EP-0,294,879). An enzyme obtained from aculture of Bacillus polymyxa is also described in U.S. Pat. No.3,812,012.

Surprisingly, it was found that cultures of Escherichia coli (K5),strain SEBR 3282, produce under certain environmental conditions anenzyme which fragments N-acetylheparosan during culture in a fermenter.No less surprisingly, it was also found that the N-acetylheparosanfragments lie around a peak of molecular mass of approximately 5000 Da,representing, apart from a few disaccharide units, an aggregate ofapproximately 70% of the product.

Lastly, again surprisingly, it was found that a suitably solubilisedpreparation of this enzyme made it possible to obtain fragments ofhigher molecular mass than the fragments obtained spontaneously, that isto say with the non-solubilised enzyme. In effect, solubilised enzymepreparations enable the fragmentation to be varied, and fragments to beobtained, the majority of which have a molecular mass at least 1000 to3000 Da higher than the molecular mass of the fragments obtainedspontaneously.

Thus, the subject of the present invention is an enzyme which makes itpossible to vary the fragmentation of a high molecular massN-acetylheparosan, and to obtain N-acetylheparosan fragments of desiredmolecular mass in very good yield. The enzyme of the present inventionis hence useful for preparing low molecular mass N-acetylheparosans froma high molecular mass N-acetylheparosan, for the following reasons:

high molecular mass N-acetylheparosan is obtained on inexpensivesynthetic medium in yields higher than those using a complex medium,such a medium being necessary for obtaining a low molecular massN-acetylheparosan;

high molecular mass N-acetylheparosan is technically easier to handlethan that of low molecular mass;

in vitro fragmentation separates the production phase from that offragmentation, enabling the two phases to be controlled and optimisedwhile affording great latitude with regard to the characteristics of theproduct sought, for example with regard to its molecular mass;

the enzyme and N-acetylheparosan are especially stable, and this allowsmultiple recyclings which may be necessary when carrying out dynamicprocesses, during which the processes of fragmentation and fractionationare carried out simultaneously or successively, while procuring freedomfrom the time constraints linked to the use of a bioreactor.

More specifically, the enzyme of the present invention is characterisedin that:

it is obtained from an Escherichia coli (K5) strain, Escherichia colistrain SEBR 3282, or from a spontaneous or induced mutant of thisstrain,

its molecular mass is between 62,000 and 70,000 Da, and morespecifically between 65,000 Da and 66,000 Da (±1500 DA),

its isoelectric point lies in the pH range between 4.7 and 5.4 pH units,and is more specifically 5.1 pH units,

it is an eliminase, and more specifically an endo-β-eliminase.

The enzyme of the present invention is also characterised in that itacts in the following manner:

it is of membrane origin,

its temperature of optimal functioning (maximum activity) is in theregion of 37° C., and the temperature at which it is inactivated isapproximately 60° C.,

the optimal pH range for its functioning lies between the values pH 6and pH 7, and more specifically between pH 6.6 and pH 6.8,

for its functioning, the optimal range of concentration of monovalent ordivalent ions lies in the vicinity of 0.2M, and more specifically 0.15Mfor divalent ions and 0.25M for monovalent ions.

Furthermore, the enzyme of the invention is characterised in that:

it does not permit fission of N-acetylheparosan below a certain size,which size corresponds to a molecular mass of approximately 1000 to 1500Da,

it is capable of acting on high molecular mass N-acetylheparosan and offragmenting it in the absence of bacterial particles in vitro.

The enzyme which is the subject of the present invention is an enzymeobtained from a culture of Escherichia coli, in particular Escherichiacoli SEBR 3282. This strain is a strain derived from the strain Bi8337-41 (O10:K5:H4) ATCC 23506 (described by D. S. Gupta et al FEMSMicrobiology Letters, 1982, 14, 75-78 and W. Vann Eur. J. Biochem. 1981,116, 359-364).

The Escherichia coli (K5) strain SEBR 3282 responds positively to thetyping test with the specific phage K5 according to the method of B.Kaiser et al (J. Clin. Microbiol., (1984), 19, 2, 264-266). Hence it isindeed an Escherichia coli (K5) strain. This strain was deposited withthe CNCM of the Pasteur Institute, Paris, France, under No. I-1013. Itis also possible to isolate this enzyme from a mutant, eitherspontaneous or induced, of Escherichia coli strain SEBR 3282, oralternatively with other suitable Escherichia coli (K5) strains, and inparticular with the strain Bi 8337-41 (O10:K5:H4) ATCC 23506. It is alsopossible to envisage obtaining this enzyme with the strain Bi 626-42(012:K5:NM) ATCC 23508.

The molecular mass of this enzyme, assessed by exclusion chromatography,is between 62,000 and 70,000 Da, and it is, more specifically,approximately 65,000 Da to 66,000 Da (±1500 Da).

The isoelectric point of the enzyme which is the subject of the presentinvention lies at a pH of 4.7-5.4, and is more specifically 5.1 pHunits.

The enzyme of the invention enables N-acetylheparosan to be fragmented,and fragments to be obtained containing at the non-reducing end aglucuronic acid residue having a double bond between carbons 4 and 5(elimination of the OH group). Such enzymes do not involve water in thechemical reaction in question, and are termed eliminase type. The enzymewhich is the subject of the present invention is hence an eliminase, andin particular an endo-β-eliminase.

The prefix "endo" is used to indicate that the enzyme which is thesubject of the present invention is capable of fragmentingN-acetylheparosan at a considerable distance from the end of themolecule. More especially, the enzyme of the present invention does notpermit fission of N-acetylheparosan below a certain size, whichcorresponds to a molecular mass of approximately 1000 Da to 1500 Da.Thus, the enzyme which is the subject of the present invention, during atotal fragmentation of a high molecular mass N-acetylheparosan, makes itpossible to obtain N-acetylheparosan fragments of molecular massesbetween 1000 Da and 10,000 Da, and more especially to obtain fragmentshaving molecular masses grouped together comprising 70% in the region of5000 Da.

The high molecular mass N-acetylheparosan which can be fragmented by theenzyme which is the subject of the present invention may be obtained asdescribed by W. F. Vann et al, Eur. J. Biochem, 1981, 116, 359-364, andin Patent Application EP-A-0,333,243, or alternatively by fermentationof the same strain Escherichia coli (K5) SEBR 3282 used for thepreparation of the enzyme of the present invention.

By the action of the enzyme of the present invention, high molecularmass N-acetylheparosan is fragmented into fragments having a predominantmolecular mass of approximately 5000 Da. The term "predominant molecularmass" is understood to mean the molecular mass which corresponds to themaximum (peak) of the chromatographic profile obtained on determinationof the molecular mass of the N-acetylheparosan by gel permeationchromatography (GPC). The fragments thereby obtained possess at thenon-reducing end a glucuronic unit having a double bond between carbons4 and 5.

This enzyme makes it possible, in particular, to obtain at the end ofculturing, from a high molecular mass N-acetylheparosan, a majority offragments having identical sizes which correspond to a molecular mass ofapproximately 5000 Da. Hence the enzyme of the invention must be capableof interacting with the N-acetylheparosan macromolecule at a givendistance from the end of this macromolecule. It is hence anendo-eliminase which enables a majority of fragments consisting of anidentical number ofβ-D-glucuronyl-(1→4)-N-acetyl-α-D-glucosaminyl-(1.fwdarw.4) units to beobtained.

Monitoring of the spontaneous fragmentation of N-acetylheparosan duringculture in a complex medium also brings to light a phenomenon of mildlysis which makes it difficult to foresee where the enzyme in questionis localised in the cell: cytoplasm, periplasm, membrane oralternatively extracelluar culture medium.

The methods employed to localise the endo-β-eliminase are defined andcollated in Scheme 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing in vivo fragmentation of different classes ofN-acetylheparosans in a fermenter at different times.

FIG. 2 is a graph showing the relative abundance of the differentclasses of N-acetylheparosans as a function of time.

FIG. 3 is an in vitro demonstration of the endo-β-eliminase activity ofa high molecular mass N-acetylheparosan.

FIG. 4 shows the determination of the pH of the eliminase enzyme usingchromatofocusing.

FIG. 5a shows a graph of molecular mass of the eliminase using gelpermeation chromatography.

FIG. 5b shows a graph of molecular mass of the eliminase using achromatography column.

FIG. 6 shows Scheme 1 which collates the methods used to localize theendo-β-eliminase.

It is observed in FIG. 6 that:

the supernatant of the suspensions of culture of Escherichia coli (K5)strain SEBR 3282 on "complex medium" contains small amounts ofendo-β-eliminase;

the major part of the enzymatic activity lies in the crude bacterialpellets;

the lipid membrane preparations obtained from the lysis supernatants ofthe washed pellets contain the major part of the β-eliminase typeactivity.

Consequently:

the enzyme is originally membrane-bound and, without furthermanipulation, the major part of the activity occurs in the lipidmembranes of the bacteria;

during culture in liquid medium, and especially with stirring, as wellas under conditions of lysis of the bacterial particles, a part of theactivity may occur and be exerted outside the bacterial particles;

it is not necessary to concentrate the enzyme; to obtain it, it sufficesto isolate lipid membranes.

The enzyme which is the subject of the present invention is hence anenzyme of membrane origin, the major part of the activity occurring inlipid membranes of the bacteria. However, during culture in liquidmedium, and especially with stirring, as well as under conditions oflysis of the bacterial particles, a part of the activity may occur andbe exerted outside the bacterial particles.

The solubilisation of the enzyme of the present invention wasestablished using different detergents according to known techniques(Biotechnology and Applied Biochemistry, 1990, 12, 599-620), startingwith a membrane pellet.

Thus, for example, enzyme preparations solubilised with detergents suchas Triton X-100®, Triton X-114®, DOC, NP-40®, Tween 80®, all at aconcentration of 2%, and 0.1M guanidine hydrochloride are active. Theenzyme may also be obtained in solubilised form by subjecting the crudebacterial pellet to an alkaline lysis.

The determination of the isoelectric point (pHi) of the enzyme wasperformed according to the "chromatofocusing" method, in a pH rangebetween 3.5 and 9. The pHi of the enzyme thus determined lies between pH4.7 and PH 5.4, more specifically at 5.1 pH units.

The molecular mass of the enzyme of the present invention was determinedby gel permeation chromatography (GPC), and lies between 62,000 and70,000, the protein peak being at approximately 65,000-66,000 Pa (±1500Da).

The enzyme of the present invention, in the form of a membranepreparation, was also subjected to tests to study the environmentalfactors that exert an influence on its activity. Thus, the influence oftemperature, pH, monovalent ions, in particular the Na⁺ ion, anddivalent ions, in particular the Ca²⁺ ion, is known. The Michaelisconstant was also determined, and the stability of the enzyme undercertain conditions was observed.

The optimal temperature of functioning of the enzyme (maximal activity)which is the subject of the present invention is in the region of 37° C.The inactivation temperature of the enzyme is 60° C., and at atemperature of 20° C. the enzyme retains 40% of its activity incomparison to that at 37° C.

The optimal pH range of functioning of the enzyme which is the subjectof the present invention lies between the values pH 6 and pH 7, and ismore specifically pH 6.6 to pH 6.8.

Moreover, the optimal range of concentration of monovalent ions, andmore especially sodium ions, for the functioning of the enzyme which isthe subject of the present invention lies in the vicinity of 0.2M, andmore specifically at approximately 0.25M.

For divalent ions, the optimal concentration range for the functioningof the enzyme also lies in the vicinity of 0.2M, and more specificallyat approximately 0.15M. In effect, when the enzyme is used dissolved in0.2M calcium chloride, an approximately 100% activation is observedrelative to the control without Ca²⁺ ion.

The invention also relates to a process for production of the enzymewhich is the subject of the present invention, and in particular theproduction of preparations containing this enzyme from a culture ofEscherichia coli (K5) strain SEBR 3282, or from a mutant, eitherspontaneous or induced, of the strain SEBR 3282. However, it is alsopossible to obtain this enzyme with other suitable Escherichia coli (K5)strains, for example with the strain Bi 8337-41 (010:K5:H4) ATCC 23506.It is also possible to envisage obtaining this enzyme with Escherichiacoli (K5) strain Bi 626-42 (012:K5:NM) ATCC 23508.

More especially, the invention also relates to a process for productionof the enzyme which is the subject of the present invention, in the formof crude bacterial pellets containing it, characterised in thatculturing of Escherichia coli (K5) SEBR 3282 or of a spontaneous orinduced mutant of this strain, or of another suitable Escherichia coli(K5) strain, is performed in a culture medium favourable to theformation of this enzyme, and in that the pellet is isolated from theculture by centrifugation, for example at 10,000×g.

The invention also relates to a preparation containing the enzyme whichis the subject of the present invention, consisting of a crude bacterialpellet obtained from a culture of Escherichia coli (K5) SEBR 3282 or ofa spontaneous or induced mutant of this strain, containing this enzyme.

The invention also relates to a process for production of the enzymewhich is the subject of the present invention, in the form of crudelysates containing it, characterised in that culturing of Escherichiacoli (KS) SEBR 3282 or of a spontaneous or induced mutant of this strainis performed in a culture medium favourable to the formation of thisenzyme, the pellet is isolated from the culture and this pellet issubjected to a lysis.

The preparations containing the enzyme which is the subject of thepresent invention, consisting of a crude lysate obtained by lysis of acrude bacterial pellet containing this enzyme, also form part of thepresent invention.

The invention also relates to a preparation containing the enzyme whichis the subject of the present invention, consisting of a membranepreparation obtained by lysis of a bacterial pellet containing thisenzyme, and then by separation of the membranes from the lysate of thebacterial pellet.

The invention also relates to the preparations containing the enzyme insolubilised form.

These preparations may be obtained by solubilising the enzyme containedin a membrane preparation using anionic or cationic detergents.

They may also be obtained by subjecting a crude bacterial pelletcontaining this enzyme to a partial lysis in an alkaline medium.

The term "preparation containing the enzyme" clearly includes thepreparations of the enzyme in the form of crude bacterial pellets, aswell as the crude lysates obtained from a culture of Escherichia coli(K5) strain SEBR 3282, or with a mutant, either spontaneous or induced,of the strain.

The term "preparation containing the enzyme" also includes the membranepreparations obtained from a crude lysate containing the enzyme which isthe subject of the present invention.

The term "preparation containing the enzyme" also includes thesolubilised enzyme preparations obtained with membrane preparationscontaining the enzyme which is the subject of the present invention, aswell as the solubilised enzyme preparations obtained after alkalinelysis of the crude bacterial pellets containing the enzyme which is thesubject of the present invention.

The term "preparation containing the enzyme" also includes any otherpreparation containing the enzyme, alone or in combination with anotherenzyme or organic or inorganic substances.

To obtain a crude bacterial pellet containing the enzyme which is thesubject of the present invention, the pellet of the suspension of anEscherichia coli (K5) strain SEBR 3282 culture performed underenvironmental conditions favourable to the formation of the enzyme whichis the subject of the present invention is used. This pellet is isolatedfrom the suspension of an Escherichia coli (K5) strain SEBR 3282 cultureby standard methods, for example by centrifugation.

In effect, an eliminase type activity is observed, during culturing ofEscherichia coli (K5) strain SEBR 3282, only under certain conditions:

1--the culture medium must contain an inducer of the synthesis of theenzyme, which can be either complex like yeast extract, or pure likeN-acetylglucosamine;

2--the culture medium must, in addition, contain an amino acid mixturesuch as, for example, casein hydrolysate (HY-CASE SF -Sheffield® USA),or alternatively a mixture of synthetic amino acids made from pure aminoacids as described in the formulation of medium E;

3--the carbon source used can be glucose or glycerol. The lattercompound does not, however, enable significant eliminase activity to beobtained.

To observe maximal eliminase activity, several factors must henceparticipate synergistically:

actual inducing factors;

synthesis-regulating factors such as the nitrogen and carbon sources;

environmental factors that influence the activity of the enzyme itselfor of its interaction with the substrate, such as monovalent or divalentions, temperature, pH or the like.

Moreover, the kinetics of eliminase activity during culture shows thateither the synthesis of the enzyme takes place mainly during thepost-exponential phase of culture, or the persisting enzyme is activatedduring this phase of culture.

It is hence apparent that induction of the enzyme may be sought in arational manner using synthetic media which simultaneously fulfil theconditions described above.

Medium E, the composition of which is given in Table I below, enablesthe formulation of efficacious culture media free from complex startingmaterials of animal origin to be envisaged.

                  TABLE I                                                         ______________________________________                                        Composition and preparation of medium E                                       MEDIUM E                                                                      Medium E is obtained by combining the following two                           sterile solutions 1 and 2:                                                    ______________________________________                                        Solution No. 1                                                                In 700 ml of ultrapurified water, dissolve in order:                          Complexing agent: N-[Tris(hydroxymethyl)methyl]-                                                         360     mg                                         glycine (Tricine marketed by Fluka ®)                                     K.sub.2 HPO.sub.4          790     mg                                         M.sub.g Cl.sub.2.6H.sub.2 O                                                                              620     mg                                         K.sub.2 SO.sub.4           610     mg                                         FeSO.sub.4.7H.sub.2 O      25      mg                                         CaCl.sub.2.2H.sub.2 O      2       mg                                         NaCl                       500     mg                                         KCl                        5000    mg                                         KI                         10      mg                                         Yeast extract              18000   mg                                         Heat gently, adjust the pH to 7.4, then make the volume to 750 ml             with ultrapurified water and autoclave for 30 minutes at 120° C.       Solution No. 2                                                                Dissolve in 100 ml:                                                           Glutamic acid              17.8    g                                          Proline                    1.6     g                                          Methionine                 0.4     g                                          Glycine                    0.75    g                                          Arginine                   1.25    g                                          Cysteine                   0.20    g                                          K.sub.2 HPO.sub.4          1.56    g                                          Solution of trace elements 1       ml                                         (see Table II below)                                                          Glucose                    18      g                                          Adjust the pH to 7.4 with KOH. Make the volume to 167 ml with                 ultrapurified water and perform a sterilising filtration                      through a 0.2 μm membrane.                                                 ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        Preparation of the solution of trace elements                                 in 800 ml of ultrapurified water, dissolve (in order):                        ______________________________________                                        H.sub.3 BO.sub.3                                                                              500          mg                                               Na.sub.2 MoO.sub.4,2H.sub.2 O                                                                 1930         mg                                               CoCl.sub.2.6H.sub.2 O                                                                         11850        mg                                               CuSO.sub.4.5H.sub.2 O                                                                         25           mg                                               ZnSO.sub.4.7H.sub.2 O                                                                         2000         mg                                               AlCl.sub.3.6H.sub.2 O                                                                         2410         mg                                               Add 100 ml of hydrochloric acid of density                                    1.19 and make to 1000 ml with ultrapurified water.                            ______________________________________                                    

As a culture medium, there may also be mentioned, as a non-exclusiveexample, a complex medium as described in the present invention, and inparticular "medium D" for preculture of Escherichia coli (K5) strainSEBR 3282, and "medium C" for culture of this preculture.

To obtain a crude lysate containing the enzyme which is the subject ofthe present invention, the pellet of the suspension of an Escherichiacoli (K5) strain SEBR 3282 culture set up under environmental conditionsfavourable to the formation of the enzyme which is the subject of thepresent invention is used, and more especially the crude bacterialpellets containing the enzyme. In effect, at the end of this culturing,the pellet of the suspension of the strain SEBR 3282 culture isrecovered by centrifugation and then lysed, for example by addition oflysozyme. This crude lysate thereby obtained may be used directly.

The membrane preparations containing the enzyme which is the subject ofthe present invention are obtained from the crude lysate by successivecentrifugation enabling the membrane pellets to be isolated, whichpellets are then solubilised, either in ultrapurified water or insuitable buffers such as, for example, Tris-HCl buffer, pH 7.2. It isalso possible to use aqueous solutions containing monovalent or divalentions, in particular Na⁺ or Ca²⁺ ions.

The solubilised enzyme preparations are obtained from membranepreparations containing this enzyme. In effect, the enzyme which is thesubject of the present invention may be solubilised from membranepreparations and obtained in active form.

In order to solubilise it, detergents, salts, enzymes or any other meansknown to a person skilled in the art may be used. In effect, the enzymewhich is the subject of the present invention may be solubilised fromlipid preparations using certain detergents without being denatured.

As detergents, nonionic detergents may be used more especially. Asexamples, Triton X-100®, Triton X-114®, deoxycholate (DOC), NP-40®,Tween 80® and 0.1M guanidine HCl may be mentioned. However,demonstration of the enzymatic activity of the enzyme which is thesubject of the present invention necessitates removal of the detergentresidues using, for example, specific, commercially available affinitycolumns.

The enzyme which forms the subject of the present invention may also besolubilised in an undenatured manner without making use of detergents,by partial lysis in an alkaline medium. As an alkaline medium (pHapproximately 11), solutions of strong inorganic bases such as sodiumhydroxide or potassium hydroxide may be used. Potassium hydroxide ispreferred.

The invention also relates to processes for fragmentation of highmolecular mass N-acetylheparosans employing preparations of the enzymewhich is the subject of the present invention.

These processes can employ the enzyme which is the subject of thepresent invention either in culture media in vivo during the productionof N-acetylheparosans, or in vitro in the case where the phase ofproduction of N-acetylheparosan is separated from that of fragmentation.In the latter case, the preparations of the enzyme which is the subjectof the present invention are used only in the fragmentation phase.

The enzymatic reaction is performed at between pH 4.0 and pH 9.0, andpreferably between pH 6.4 and pH 7.4.

The in vivo demonstration of the enzyme was accomplished by the inducedfragmentation of a quantity of high molecular mass N-acetylheparosanadded to a culture suspension possessing eliminase activity.

It was confirmed that the enzyme thereby obtained cuts high molecularmass N-acetylheparosan so as to obtain an N-acetylheparosan composedpredominantly of fragments of molecular mass 5000 Da. Hence thephenomenon does not represent a halting of the synthesis of highmolecular mass N-acetylheparosan by the bacterium, but a subsequentmodification of the polymer.

It was found, surprisingly, that the molecular mass of theN-acetylheparosan obtained by fragmentation performed with the enzyme ofthe present invention may be varied by using a solibilised enzymepreparation or alternatively by carrying out the enzymatic reaction inthe presence of cations, in particular Na⁺ ions.

Thus, according to a further aspect, the present invention relates to aprocess for the preparation of N-acetylheparosan having molecular massesgrouped together, lying, in particular, between 2000 and 10,000 Da,characterised in that high molecular mass N-acetylheparosan(polysaccharide K5) is treated with the enzyme of the present inventionin the presence of a solution of sodium chloride having, in particular,a molarity of 0.2 to 0.5M.

The term "molecular masses grouped together between 2000 and 10,000 Da",as used here, relates to the different N-acetylheparosans having amolecular mass with a fairly narrow distribution curve, for example 1000to 3000 Da with predominant molecular mass at 2000 Da, from 3200 to 4000Da with predominant molecular mass at 3600 Da, from 3900 to 4700 Da withpredominant molecular mass at 4300 Da, from 6000 to 8000 Da withpredominant molecular mass at 7000 Da, and from 8000 to 10,000 Da withpredominant molecular mass at 9200 Da. It is understood that thesemolecular masses refer to the majority of the constituents, namelybetween approximately 50% and approximately 90%. The molecular mass ofthe products thereby obtained depends, in particular, on the molarity ofthe medium with respect to sodium chloride in the reaction medium, aswell as on the form, solubilised or otherwise, of the enzyme.

The process of the present invention may also be performed by bindingthe high molecular mass N-acetylheparosan to a column of ion exchangeresin and treating the product bound to the column with a solution ofthe enzyme of the present invention.

On eluting with a solution containing 0.2 to 0.5M sodium chloride,N-acetylheparosan having the desired molecular mass, depending on themolarity with respect to sodium chloride of the solution used, isobtained. For this variant, anion exchange resins, for example a QSepharose® type resin or other equivalent resins, may be used as theresin.

Moreover, in view of the fact that the enzyme which is the subject ofthe present invention is active over a wide range of sodium chlorideconcentrations, it is possible to envisage the use of the enzyme inprocesses which permit a fragmentation and a simultaneous fractionationof the N-acetylheparosan bound to the column.

The invention also relates to processes for preparation of low molecularmass N-acetylheparosans using the enzyme which is the subject of thepresent invention in immobilised form.

The invention also relates to the use of the enzyme for thefragmentation of high molecular mass N-acetylheparosans.

For the preparation of low molecular mass N-acetylheparosans, it is alsopossible to envisage a process that combines a step of fragmentation ofa high molecular mass N-acetylheparosan with the enzyme which is thesubject of the present invention, and an alcohol fractionation step oran ultrafiltration step.

The examples which follow illustrate the invention without, however,limiting it. In the PREPARATION and in the EXAMPLES, trade names whosemeanings are clarified below have been used:

Triton X-100®: polyethylene glycol tert-octylphenyl ether (Rohm & HassCo.)

Triton X-114®: polyethylene glycol tert-octylphenyl ether (Rohm & HassCo.)

DOC: deoxycholic acid sodium salt

NP-40®: Nonidet® P-40, ethylphenylpolyethylene glycol (Shell)

Tris-HCl: Tris(hydroxymethyl)aminomethane hydrochloride

Tween 80®: polyoxyethylene sorbitan monooleate (Atlas Chem. Ind. Inc)

Struktol J 673®: antifoam of Schill & Seilacher--Germany/Hamburg

PREPARATION

N-Acetylheparosan predominantly of high molecular mass

400 ml of medium B of composition specified in Table III below, areinoculated with Escherichia coli (K5) strain SEBR 3282 deposited withthe CNCM of the Pasteur Institute, Paris--France, under No. I-1013, andthe suspension is incubated with stirring for 2 h at 37° C. Thepreculture obtained is then transferred to an 18.5-l fermentercontaining 11 ml of medium A, of composition also specified in Table IIIbelow, and the suspension is incubated for 6 h 30 minutes at 37° C. andpH 7.2, the oxygen partial pressure being maintained at 40 mmHg byregulating the injection of air (up to 20 l/minute) and with stirring.Glycerol is then added by introducing in continuous fashion a sterilesolution containing 500 g/l of glycerol at the rate of 18 g/h for 16 to17 hours.

Culturing is continued under the same temperature, pH and oxygen partialpressure conditions until virtually all the glycerol has been consumed.Monitoring of the optical density (OD) at λ=600 nm of the culturesuspension after the addition of glycerol has been completed shows astationary state or state of mild lysis until culturing is halted at28-30 h of age in the fermenter.

                  TABLE III                                                       ______________________________________                                        Composition and preparation of medium A and medium B                          ______________________________________                                        MEDIUM A                                                                      In 900 ml of ultrapurified water, dissolve in order:                          NTA (nitrilotriacetic acid)*                                                                           1000    mg                                           K.sub.2 HPO.sub.4        790     mg                                           Glutamic acid            11000   mg                                           MgCl.sub.2.6H.sub.2 O    500     mg                                           K.sub.2 SO.sub.4         450     mg                                           FeSO.sub.4.7H.sub.2 O    18      mg                                           CaCl.sub.2.2H.sub.2 O    2       mg                                           NaCl                     500     mg                                           KCl                      5000    mg                                           Solution of trace elements (see Table II)                                                              1       ml                                           Glycerol                 10000   mg                                           Adjust the pH to 7.2 with concentrated potassium hydroxide of                 density 1.38, and make to 1000 ml with ultrapurified water.                   Perform a sterilising filtration through a 0.2 μm membrane.                Glycerol solution                                                             Dissolve 50 g of glycerol in an appropriate quantity of ultrapurified         water and adjust the volume to 1000 ml with the same solvent.                 Perform a sterilising filtration through a 0.2 μm membrane.                The antifoam employed during fermentation is                                  Struktol J 673 ® (Schill and Seilacher).                                  MEDIUM B                                                                      The preparation of medium B is identical to that of medium                    A except that, in addition, the buffer (pH 7.2)                               3-morpholinopropanesulphonic acid (MOPS) should be added                      after addition of the antifoaming agent.                                      ______________________________________                                         *The quantity of NTA used for the preparation of medium B may be replaced     by an appropriate quantity of N[tris(hydroxymethyl)methyl]glycine (Tricin     marketed by Fluka ®), which is 360 mg.                               

The culture broth is then cooled to 25° C. 5 liters of culture arewithdrawn and centrifuged (11,000-14000×g) for 15 to 20 min. Thesupernatant is mixed with 5 liters of culture. The centrifugationoperation is repeated. The pellet is removed and the supernatant isfiltered through a 0.2 μm polycarbonate screen membrane (Nucteopore®).The filtrate obtained is concentrated using an Amicon® hollow-fibrecartridge, cut-off threshold 30,000 Da, or equivalent. A solutionenriched in high molecular mass N-acetylheparosan is thereby obtained.An appropriate quantity of sodium chloride is added no the solution soas to have a solution which is 0.5M with respect to NaCl, and 4 volumesof ethanol are then added. The precipitate is allowed to form for 5minutes at room temperature. The suspension is centrifuged at 5000×g for20 minutes. The centrifugation pellets are taken up in ethanol, and thesuspension obtained is stirred and left standing for 1 hour at roomtemperature. The centrifugation and suspension operations are repeated.The suspension is centrifuged again at 5000×g for 20 minutes. Thecentrifugation pellets obtained are dried in an oven under vacuum at 40°C. for 24 hours. The N-acetylheparosan obtained is a "purified highmolecular mass N-acetylheparosan". The N-acetylheparosan is then takenup in sterile water so as to have a solution of high molecular massN-acetylheparosan of concentration 28 g/l.

EXAMPLE 1

Production and characterisation of the enzyme ##STR2##

To obtain the enzyme, culturing of Escherichia coli (K5) strain SEBR3282 is performed under environmental conditions favourable to enzymeformation. In effect, it is necessary to use a complex culture medium asdescribed below. However, other equivalent media may be used. Media Cand D, whose composition is shown in Table IV below, will be referred tohereinafter as "complex media". 400 ml of medium D are inoculated withEscherichia coli strain SEBR 3282 (deposited with the CNCM of thePasteur Institute, Paris, France, under No. I-1013), and the suspensionis incubated with stirring for 2 h at 37° C. The preculture obtained isthen transferred to an 18.5-l fermenter containing 11 l of medium C, andthe suspension is incubated for 4 h at 37° C. and at a pH equal to 7.4,the oxygen partial pressure being maintained at 40 mmHg by regulatingthe injection of air (up to 20 l/min) and with stirring. Glucose is thenadded by introducing in continuous fashion a sterile solution containing600 g/l of glucose at the rate of 250 ml/h for 8 h. Culturing iscontinued under the same temperature, pH and oxygen partial pressureconditions for 10 h after the addition of glucose has been completed.

Monitoring of the optical density (OD) at λ=600 mm of the culture mediumenables it to be asserted that there is no growth of the biomass duringthe last 12 hours of culture.

                  TABLE IV                                                        ______________________________________                                        Composition and preparation of medium C and medium D                          (COMPLEX MEDIA)                                                               ______________________________________                                        MEDIUM C                                                                      Medium C is prepared by combining the three sterile                           solutions below:                                                              Solution No. 1                                                                In 700 Ml of ultrapurified water, dissolve in order:                          Complexing agent: N-[Tris(hydroxymethyl)methyl]-                                                         360     mg                                         glycine (Tricine marketed by Fluka ®                                      FeSO.sub.4.7H.sub.2 O      280     mg                                         CaCl.sub.2.2H.sub.2 O      6.7     mg                                         MgCl.sub.2.6H.sub.2 O      1270    mg                                         K.sub.2 SO.sub.4           500     mg                                         KCl                        5000    mg                                         Casein hydrolysate (main source of amino acids)                                                          25000   mg                                         HY CASE SF ® (marketed by Sheffield)                                      Yeast extract (marketed by Difco ®)                                                                  18000   mg                                         Solution of trace elements (see Table II)                                                                1       ml                                         Antifoaming agent Struktol J673 ® (marketed by Schill and                 Seilacher):                                                                   a few drops with a Pasteur pipette.                                           Adjust the pH to 7.4 with KOH solution (d = 1.38) and                         make to 850 ml with ultrapurified water. Autoclave the                        medium for 45 minutes at 120° C.                                       Solution No. 2                                                                Dissolve 5 g of K.sub.2 HPO.sub.4, in approximately 40 ml of                  ultrapurified                                                                 water and then adjust to 50 ml with the same solvent. Filter                  the solution obtained through a filter of porosity 0.2 μm.                 Solution No. 3                                                                Dissolve 20.7 g of glucose in an appropriate quantity of ultrapurified        water and adjust the volume to 100 ml with the same solvent.                  Autoclave at 110° C. for 30 minutes.                                   MEDIUM D                                                                      The preparation of medium D is identical to that                              of medium C except that, in addition, 20 g of pH 7.2                          buffer (3-morpholinopropanesulphonic acid) should be                          added after addition of the antifoaming agent.                                ______________________________________                                    

An Escherichia coli (K5) culture containing the enzyme which is thesubject of the invention is thereby obtained. ##STR3##

The in vivo demonstration of the enzyme was accomplished by the inducedfragmentation of a quantity of high molecular mass N-acetylheparosanadded to a culture suspension possessing eliminase activity.

The aqueous solution of high molecular mass N-acetylheparosan obtainedin the preceding stage is added in an 18.5-liter fermenter to anEscherichia coli (K5) strain SEBR 3282 culture containing the enzymewhen the culture has reached the plateau. This is demonstrated by themonitoring of the OD at λ=600 nm (1--Production of an Escherichia coli(K5) SEBR 3282 culture containing the enzyme).

Samples of culture suspension were taken at 3, 5, 7, 9.75 and 13.5 hoursafter addition of exogenous N-acetylheparosan, as well as immediatelyafter this addition.

Step A

Determination of the distribution of molecular masses ofN-acetylheparosans during the fermentation

The determination of the distribution of molecular masses of theN-acetylheparosans contained in the different samples was performed byexclusion HPLC.

a--Exclusion HPLC operating conditions

Column: TSK G 3000 SW® (LKB) 7.5×300 mm consisting of silica beads 10 μmin diameter and of porosity 250 Å.

Eluent: 0.5M aqueous sodium sulphate solution, filtered through 0.2 μmand degassed.

Flow rate: 1 ml/minute

UV detection at λ=205 nm

calibration is performed using a series of oligosaccharides derived fromheparin (10 mg), f the following molecular masses (Da): 1320, 1880,2440, 3410, 4000, 4540, 5000, 5370, 5730, 6150, 6670, 7540, 8660, 10090,11560, 12950, 14810, 17390, 22670.

b--Sample preparation

The starting material is a sample taken from the culture suspension in afermenter. Centrifuge at 7000×g for 2 minutes. Recover the supernatantand filter it through a 0.2 μm membrane, precipitate the filtrate byadding 4 volumes of absolute ethanol, equivalent to a final 80% ofethanol, vortex and wait 5 minutes. Centrifuge at 4000×g for 5 minutes,remove the supernatant, take the pellet up in ultrapurified water to theinitial volume, vortex and dialyse using a Spectra-Por® sac (Spectrum)of cut-off threshold 10,000 Da overnight against ultrapurified water.

With magnetic stirring at 4° C., add concentrated NaCl solution suchthat the final concentration of the solution containing theN-acetylheparosan is 0.5M. Precipitate by adding 4 volumes of absoluteethanol per volume of salted dialysate, and vortex. Wait 5 minutes,centrifuge at 4000×g for 5 minutes, discard the supernatant, take thepellet up in 25 mM piperazine buffer, pH 3.5 so as to obtain aconcentration lying between 5 and 10 g of N-acetylheparosan/liter, andvortex strongly.

Deposit the equivalent of 1 mg of N-acetylheparosan on a column of QSepharose® resin previously equilibrated with the piperazine buffer, andwash the column of resin with 2 volumes of piperazine buffer. Wash with4-5 volumes of ultrapurified water, elute with 2 volumes of 0.5M NaCl,collect the eluate and precipitate with absolute ethanol as above.

Centrifuge an 4000×g for 5 minutes, and take the pellet up inultrapurified water so as to obtain a concentration lying between 5 and10 g/l of N-acetylheparosan.

This solution is ready for the determination of the distribution ofmolecular masses by exclusion chromatography.

The results corresponding to the samples analysed are given in the formof chromatographic profiles in FIG. 1.

From these data, the N-acetylheparosan fragments were divided into threeclasses:

Fragments having a molecular mass of greater than 100,000 Da

Fragments having a molecular mass of between 5000 and 100,000 Da

Fragments having a molecular mass equal to approximately 5000 Da.

The relative abundance of 3 classes of fragments in the fermenter as afunction of time is shown in FIG. 2.

Examination of FIG. 1 permits the following conclusions:

The high molecular mass N-acetylheparosan of the culture and that whichwas added thereto gradually change into N-acetylheparosan predominantlyof low molecular weight.

Examination of FIG. 2 permits the following conclusions:

There are transient steps in which N-acetylheparosan of intermediatemolecular mass is detected, that is to say the whole range of molecularmasses is represented at the same time. However, after 24 hours, thehigh molecular mass N-acetylheparosan introduced into the fermenter isfragmented virtually completely (80%) into fragments having a molecularmass of approximately 5000 Da.

The fragmentation is hence performed on exogenous N-acetylheparosaneither by enzymes in solution, or in contact with membrane enzymes.

Step B

Verification of the structure of the low molecular massN-acetylheparosans obtained by NMR

The proton and carbon ¹³ C NMR spectra of the N-acetylheparosansobtained after enzymatic fragmentation are compared with those ofN-acetylheparosan which are described by W. E. Vann (Eur. J. Biochem.,1981, 116, 359-364).

A study of the spectra obtained with the low molecular mass(approximately 5000 Da) N-acetylheparosans confirms the chemicalidentity of the product with the N-acetylheparosan described by W. E.Vann. The compound comprises polymer chains consisting of repeatedβ-D-glucuronyl(1→4)-N-acetyl-α-D-glucosaminyl-(1→4) structures.

Furthermore, these same spectra enable it to be concluded that thefragments contain at the non-reducing end a glucuronic acid residuehaving a double bond between carbons 4 and 5.

The results obtained clearly indicate that the enzyme responsible forthe fragmentation is of the β-eliminase type, by virtue of the presenceof a double bond at the non-reducing end of the low molecular massN-acetylheparosan obtained, this end being a glucuronic acid residue.

Moreover, this enzyme makes it possible to obtain, at the end ofculturing, from a high molecular mass N-acetylheparosan, a majority offragments having identical sizes which correspond to a molecular mass ofapproximately 5000 Da. Hence the enzyme of the invention must be capableof interacting with the N-acetylheparosan macromolecule at a givendistance from the end of this macromolecule. This enables fragmentsconsisting of a very precise number of"β-D-glucuronyl-(1→4)-N-acetyl-α-D-glucosaminyl-(1.fwdarw.4)" units tobe obtained. The enzyme is hence an endo-β-eliminase. ##STR4##

The main steps shown in Scheme 1, which steps have made it possible toobtain preparations containing the enzyme, and in particular a crudelysate and a membrane preparation having enzymatic activity, areillustrated below.

Step A

Production of a crude lysate possessing enzymatic activity. Assessmentof its activity

a--Preparation of a crude lysate

The crude lysate possessing enzymatic activity was prepared from thepellet of an Escherichia coli (K5) strain SEBR 3282 culture suspensioncontaining the enzyme (1--Production of an Escherichia coli (K5) strainSEBR 3282 culture containing the enzyme), in the following manner:

After incubation, the culture medium is cooled to 25° C. and thensubjected to a centrifugation (31.000×g) for 15 minutes. To 200 g of wetpellets, 400 ml of 50 nM Tris-HCl buffer, pH 8 and an appropriatequantity of EDTA so as to obtain a final concentration of 10 mM areadded. 100 mg of lysozyme are then added.

The enzymanic reaction performed with lysozyme is carried out at roomtemperature, the pH being maintained at 8.0. When the mixture ishomogeneous, it is placed at 4° C. overnight. After this time interval,the pH is readjusted to 7.5 and MgCl₂ is added to a final concentrationof 20 mM, followed by DNase I®, 15 mg in total

b--Demonstration of the fragmentation produced by the crude lysate

Using the crude lysate described above, an in vitro fragmentation of ahigh molecular mass N-acetylheparosan was observed using the followingprocedure:

The pH of the crude lysate is adjusted to 6.8. 600 μl are withdrawn, and1 mg of purified high molecular mass N-acetylheparosan, as described inthe PREPARATION is added thereto. The mixture is then incubated at 37°C. for 24 hours. When the reaction is complete, the distribution ofmolecular masses of the N-acetylheparosan obtained is carried out.

The determination of the distribution of molecular masses of the sampleswas performed by exclusion HPLC as described above (2--In vivodemonstration of the enzyme in a fermenter--Step A).

A high molecular mass N-acetylheparosan control without lysate was alsotreated under the same conditions. Comparison of the chromatographicprofiles obtained demonstrates that no fragmentation of the control hastaken place (FIG. 3--broken lines). It is still a high molecular massN-acetylheparosan composed of constituents having molecular massesranging from 10,000 to 100,000 Da.

In contrast, a fragmentation is observed when the high molecular massN-acetylheparosan is treated with the crude lysate. On thechromatographic profile, fragments are observed having a molecular massof between approximately 4000 and 8000 Da, with a peak amassingfragments of approximately 5000 Da corresponding to the major part ofthe final product (FIG. 3--continuous lines).

The β-eliminase type enzymatic activity of the strain SEBR 3282 mayhence be exploited in the absence of bacterial particles, in vitro,using a simple crude lysate. It is also highly surprising that thefragments are not split below a certain size, in contrast, for example,to what is obtained with the phage lyase specific for the strainEscherichia coli (K5), which effects the total disappearance of the5000-Da fragments in favour of much smaller entities (Gupta D., Jann Band Jann K; FEMS Microbiology Letters 1983 16, p. 13-17).

Step B

Production of a membrane preparation possessing enzymaticactivity--Assessment of its activity

The crude lysate obtained in the preceding step is subjected to acentrifugation at 15,000×g for 60 minutes. The pellet is removed and thesupernatant is centrifuged at 105,000×g for 60 minutes. The pelletcontaining the bacterial membranes is isolated and taken up in theinitial volume with purified water.

Demonstration of the eliminase activity of this membrane preparation isperformed as described in the preceding step, incubating an aliquot ofthis preparation brought into contact with high molecular massN-acetylheparosan at a pH in the region of neutrality and at atemperature of 37° C. After one night, a sample is withdrawn and theassessment of the distribution of its molecular mass is performed byHPLC using the method quoted above (2--In vivo demonstration of theenzyme in a fermenter--Step A). The results obtained show that theendo-β-eliminase enzymatic activity occurs in the bacterial membranes.This does not rule out, as indicated in Scheme 1, the possibility ofdetecting an activity in the supernatant of the culture suspension,obtained after low-speed centrifugation (15,000×g). In effect, a partiallysis of the bacteria takes place spontaneously, after the plateauphase, within the culture suspension. ##STR5##

4.1--Solubilisation using detergents

As a result of the techniques for assay of enzymatic activity whichnecessitate a functional enzyme, the detergents in question were chosenpredominantly from nonionic and non-denaturing detergents (Schutte H,Kula M. R; Biotechnology and Applied Biochemistry (1990), 12, 559-620).

Solubilisation of the enzyme in membrane form was performed using amembrane pellet as described above (3--Localisation of theenzyme--Production of preparations containing this enzyme--Step B).

The membrane pellets are taken up in a solution of detergents of thesame volume as the initial crude lysate, and the mixture is then left incontact overnight at 4° C. It is then subjected to a centrifugation at105,000×g for 1 hour. The supernatant containing the proteins isrecovered, dialysed and passed through an affinity column fordetergents, according to the directions of the supplier.

Demonstration of the activity of the different preparations containingthe enzyme in soluble form was performed by incubating an aliquot of thehigh molecular mass preparations at a pH in the region of neutrality andat a temperature of 37° C.

After 6 days of incubation, samples are withdrawn and the distributionof molecular masses of the N-acetylheparosans obtained is assessed byHPLC according to the method described above. The results of this studyare collated in Table V.

A portion of the membrane pellet, taken up in 50 nM Tris-HCl buffer, pH8, served as control. The latter was treated under the conditions quotedabove.

Assay of total proteins was performed using "BCA protein assay reagent"of Pierce®.

The results shown in Table V demonstrate that, apart from the sampletreated with guanidine thiocyanate, under the operating conditionsshown, the enzyme preparations solubilised with detergents are activeprovided the detergent concentration of the aqueous solution is loweredafter solubilisation of the enzyme.

In effect, the control after 6 days of incubation at 37° C. exhibitsonly a slight attack of N-acetylheparosan, of the order of 10%, whereasin the other tests, the fragmented N-acetylheparosan of low molecularmass represents approximately 90% of the initial mass, this being amaximum.

                  TABLE V                                                         ______________________________________                                                                   DEGREE                                                              MEM-      OF                                                                  BRANE     SOLUBIL-                                                            PRO-      ISATION*/                                                                              ENDO-β-                                               TEINS     MEM-     ELIMI-                                                     IN g/l IN BRANE    NASE                                                       SOLUBLE   PRO-     ACTIV-                                        TEST         FORM      TEINS    ITY                                       ______________________________________                                        1.  Tris-HCl     0.32        17%    SLIGHT                                        control                                                                   2.  Triton       0.88      44.5%    STRONG                                        x-100 ®, 2%                                                           3.  Triton       1.64      68.6%    STRONG                                        X-114 ®, 2%                                                           4.  DOC, 2%      0.86      55.1%    STRONG                                    5.  NP-40 ®, 2%                                                                            0.97      58.0%    STRONG                                    6.  GUANIDINE    2.72      90.7%    NIL                                           THIOCYANATE                                                                   4M                                                                        7.  Tween        0.57      24.1%    STRONG                                        80 ®, 2%                                                              8.  Guanidine    1.04      58.4%    STRONG                                        HCl, 0.1 M                                                                ______________________________________                                    

Another fact of interest which becomes apparent when the chromatographicprofiles are superposed is the shifting of the peak amassing theN-acetylheparosan fragments predominantly of low molecular masses of5000 Da. For the membrane enzyme (control), it lies at around 5000 Da.For the sample treated with Triton X-114®, it lies at around 6500 to7000 Da (according to heparin standard).

A study of the solubilisation of the enzyme enables it to be concludedthat:

the enzyme may be solubilised by a large number of detergents andobtained in active form;

the soluble form of the enzyme makes it possible to obtain peaksrepresenting an aggregate of a majority of N-acetylheparosan fragmentsaround a molecular mass (top of the peak) higher than that observed forthe membrane form of the enzyme.

4.2--Solubilisation by alkaline lysis

Solubilisation of the enzyme by alkaline lysis was performed using acrude bacterial pellet. The crude bacterial pellet was obtained bycentrifugation of the culture medium at 10,000×g for 10 minutes(3--Localisation of the enzyme--Production of preparations containingthis enzyme--Step A).

The bacterial pellet was frozen at -20° C. 180 g of this bacterialpellet are suspended in 100 ml of ultrapurified water, and the pH of thesuspension is adjusted to 11 using concentrated sodium hydroxidesolution. The alkaline suspension is maintained at pH 11 and at 4° C.for 2 hours. The lysate thereby obtained is ultracentrifuged at105,000×g for one hour. The supernatant is recovered, the pH is adjustedto 7 and the mixture is then dialysed at 4° C. overnight in a sac ofcut-off point 10,000 against 10 liters of ultrapurified water (waterflow rate 90-120 ml/hour). A portion of the lysate thereby obtained isstored at 4° C., and another at -80° C.

To determine the enzymatic activity, 500 μl of high molecular massN-acetylheparosan (PREPARATION) were incubated for 15 hours at 37° C.with 125 μl of the lysate obtained and stored either at 4° C. or at -80°C. Determination of the molecular mass of N-acetylheparosan obtainedafter incubation demonstrated that the activity of the lysate obtainedby alkaline lysis is strong. The storage conditions do not appear tohave an important role, since the same activity was observed with thelysate stored at 4° C. and with that stored at -80° C.

A study of the solubilisation of the enzyme enables it to be concludedthat:

solubilisation of the enzyme by alkaline lysis and ultracentrifugationenables a β-eliminase type activity to be obtained which is of the sameorder as that resulting from the action of detergents;

transitory freezing of the lysate at -80° C. does not give rise to anyloss of enzymatic activity. ##STR6##

Determination of the pHi of the eliminase is carried out by means of thechromatofocusing method. The principle of this is to create a pHgradient across a chromatography column. The proteins are then elutedwhen their charges are overall neutral (at their pHi). The pH rangetested is between pH 3.5 and pH 9.

a--Equipment used

Low pressure column: 10×30 cm (Pharmacia®)

PBE 94 gel (Pharmacia®)

Eluent: Polybuffer 96 and Polybuffer 74 (Pharmacia®)

Starting buffer for 6<pH<9:0.025M diethanolamine-HCl, pH 9.5

Starting buffer for 3.5<pH<6:0.025M histidine-HCl, pH 6.2

Pump: Gilson®.

b--Production of solubilised eliminase

4 g of wet membrane pellets are introduced into 10 ml of 0.1M Tris-HClbuffer, pH 10.5 containing 0.3M CaCl₂. The solubilisation operationtakes 2 hours at room temperature. The suspension is then centrifuged at105,000×g for 1 hour. The supernatant containing the enzyme is at aconcentration of 3 mg/ml of active proteins.

Initially, the pH range chosen lay between pH 6 and 9. More accurateresults were obtained by repeating the tests and using a pH rangebetween 4.7 and 5.4.

The enzymatic activity of the different fractions was assessed bybringing the fractions obtained during the gradient chromatography intocontact with a high molecular mass N-acetylheparosan, and determiningthe distribution of molecular masses of the N-acetylheparosan obtainedafter incubation for 60 hours at 37° C. and at pH 6.8.

The pHi of the enzyme is between pH 4.7 and pH 5.4.

Further chromatofocusing experiments were undertaken in order to definethe pHi of the enzyme more precisely. Maximal activity was found in afraction of pH 5.1. The pHi of the eliminase is consequently 5.1 (FIG.4). ##STR7##

Test 1

Determination of the molecular mass of the eliminase was performed bygel permeation chromatography (GPC). The proteins are eluted as afunction of their molecular mass, the smallest sizes emerging first.

a--Equipment used

Column: TSK G 2000 SW®, 7.5×300 mm

Eluent: 0.3M NaCl/50 mM Tris, pH 7

Flow rate: 0.3 ml/minute

Detection at 280 nm with a sensitivity of 0.2 AUFS

b--Production of solubilised eliminase.

4 g of wet membrane pellets are introduced into 10 ml of 0.1M Tris-HClbuffer, pH 10.5 containing 0.3M calcium chloride. Solubilisation takes 2hours at room temperature. The suspension is then centrifuged at105,000×g for 1 hour. The supernatant containing the enzyme is at aconcentration of 3 mg/ml of active proteins. 24 fractions collectedevery 30 seconds were obtained. The enzymatic activity of the differentfractions obtained was assessed by bringing each fraction into contactwith a high molecular mass N-acetylheparosan. After incubation for 38hours at 37° C. and at pH 7, the distribution of molecular masses of theN-acetylheparosan obtained was determined. The most active fractions lieat between 22 and 23 minutes (FIG. 5a). On the basis of the two highestpeaks with respect to eliminase activity, the molecular mass of theeliminase lies between 62,000 and 70,000 Da, and more precisely at about65,000 Da. The initial suggestion is hence that the eliminase has amolecular weight lying between 62,000 and 70,000 Da, and more especiallyat about 65,000 Da.

Test 2

In order to define the molecular mass of the enzyme more precisely, anadditional test was carried out using a chromatography column, TSK G3000 SW® column, 7.5×300 mm. This column permits a better separation ofmolecular masses of approximately 100,000 Da. The technique used and theoperating conditions are identical to those described in Test 1. Thecalibration includes molecular masses of 43,000 Da and 67,000 Da. Inthis test, it is observed that the retention times of the fractionshaving maximal activity of the enzyme correspond to molecular masses of65,000 Da to 66,000 Da (±1500 Da) (FIG. 5b). ##STR8##

For this study, a membrane preparation as described above(3--Localisation of the enzyme--Step B--Production of a membranepreparation possessing enzymatic activity. Assessment of its activity)was used. The enzymatic activity of this preparation, observed accordingto the different parameters studied, was assessed by determining thedispersion of molecular masses of a high molecular massN-acetylheparosan subjected to its action.

a--Temperature

The optimal temperature of functioning of the enzyme is in the region of37° C., its inactivation temperature is 60° C., and at 20° C. the enzymeretains 40% of its activity in comparison to that at 37° C.

b--pH

The optimal range of functioning of the enzyme lies between the valuespH 6 and pH 7. At pH 5 and at pH 7.5, the enzyme retains 40% of itsactivity in comparison to that observed in the range pH 6-7. At pH 8.5,the enzyme retains less than 10% of this activity.

c--Effect of monovalent ions (Na⁺)

The optimal range of concentration of sodium ions for the functioning ofthe enzyme lies in the vicinity of 0.2M. At this concentration ofmonovalent ions, the activity of the enzyme increases by approximately15% relative to the control. It should be noted here that sodium ionsenable the top of the peak of the N-acetylheparosan fragments to beshifted towards the high molecular masses. The membrane enzyme placedunder these conditions generates fragments of molecular massapproximately 500 Da higher than those of the control.

Some monovalent ions, especially sodium, hence enable the size of thefragments obtained at the end to be varied.

d--Effect of divalent ions (Ca²⁺)

The optimal range of concentrations of calcium ions for the functioningof the enzyme lies in the vicinity of 0.2M. With this concentration, theactivity of the enzyme increases by approximately 50% relative to thecontrol.

e--Michaelis constant

Measurement of the fragmentation reaction as a function of theconcentration of substrate (in this case high molecular massN-acetylheparosan) enables it to be established, after a Lineweaver-Burkplot:

that a straight line is obtained; the enzyme hence obeys Michaeliskinetics;

that the Michaelis constant is equal to the substrate concentration forwhich the reaction rate is half the maximal rate, and is practicallyequal to 1 g of N-acetylheparosan/liter of reaction solution;

that high substrate concentrations, for example 30 g/l, at and abovewhich the N-acetylheparosan solution begins to become viscous, do notconstitute an appreciable obstacle to the good functioning of theenzyme.

f--Stability of the enzyme

Influence of pH:

After a change to pH 10.5 at which the enzyme is inactivated, the enzymerecovers its activity once it has returned to pH values close toneutrality. The enzyme also withstands a change to pH 3.5.

Influence of temperature--Storage at 4° C.:

After storage at 4° C. for 3 months, no significant loss of activity isobserved in regard to the enzyme.

Influence of a reducing agent:

The presence of dithioerythritol (DTE) exerts a stabilising influence onthe activity of the enzyme. ##STR9##

The crude bacterial pellet, which can be obtained after centrifugationof the culture medium at 10,000×g for 10 minutes, may also be used tofragment high molecular mass N-acetylheparosan for preparatory purposes.The conditions for using crude bacterial pellets, previously induced inorder to have enzymatic activity with the object of fragmenting highmolecular mass N-acetylheparosan, were studied.

a--Temperature

The temperature of maximal enzymatic activity of the pellet is in theregion of 40° C. The pH interferes slightly with the effect oftemperature on this product.

b--pH

The optimal range of enzymatic activity of the pellet lies between thevalues pH 6.6-6.8. At pH 7.5, the enzyme retains 40% of its maximalactivity.

c--Effect of monovalent ions (Na⁺)

Among the monovalent ions studied, Na⁺, K⁺ and Li⁺ in the form of salts(chlorides), the Na⁺ ion exerts the greatest activating effect on theenzymatic activity of the crude bacterial pellet. The maximal activatingeffect is observed when the concentration of Na⁺ ions is approximately0.3M.

d--Effect of divalent ions (Ca²⁺)

Among the divalent ions studied, Ca²⁺ and Mg²⁺ in the form of salts(chlorides), Ca²⁺ ion exerts the greatest activating effect on theenzymatic activity of the crude bacterial pellet. The optimal range ofconcentration of calcium ions lies at 0.1-0.15M. Under these conditions,the activity of the enzyme is multiplied twofold.

Divalent metal ions such as Zn²⁺ and Cu²⁺ proved to be more or lessinhibitory of this enzymatic activity.

e--Michaelis constant

As a result of the characteristics of the crude preparation, the kineticstudy carried out on the enzyme linked to the bacterial particles isinterpreted with reservation. However, it was established that:

the enzymatic activity of the crude bacterial pellet shows Michaelistype behaviour, like the enzyme obtained in membrane form (membranepreparation);

the Michaelis constant under the optimal conditions (temperature, pH,Ca²⁺ ion concentration) is of the order of 2.2 to 3.2 g ofN-acetylheparosan per liter of reaction solution.

The Michaelis constant for the crude bacterial pellet is hence markedlyhigher than that found for the membrane preparation.

f--Competitive inhibitors of the enzyme/substrate interaction

It was observed that N-acetylglucosamine and glucuronic acid, both ofwhich are components of N-acetylheparosan, are competitive inhibitors ofthe enzymatic activity of the crude bacterial pellet.

g--Modification of the substrate

The carboxyl and N-acetyl groups of N-acetylheparosan were removed with1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) andsodium hydroxide in the heated state. This polymer, deprived of one orother of these groups, is no longer attacked by the enzyme.

h--Stability of the enzymatic activity of the pellet

Influence of temperature

The bacterial pellet may be stored at -20° C. or -80° C. withoutsignificant loss of its eliminase activity. ##STR10##

To study the kinetics of fragmentation of N-acetylheparosan by theenzyme contained in the crude bacterial pellet, a sample of highmolecular mass N-acetylheparosan was brought into contact with aneliminase-rich Escherichia coli (K5) SEBR 3282 bacterial pellet at 40°C. in the presence of 140 mM CaCl₂ and 50 mM bis-tris propane buffer, pH6.6. At the end of the enzymatic reaction, a fragmentation of theN-acetylheparosan of the order of 90% was observed. In effect, 90% ofthe high molecular mass N-acetylheparosan was converted to low molecularmass heparosan. The fragmented N-acetylheparosan was recovered, broughtinto contact again with a "fresh" crude bacterial pellet and incubatedagain at 37° C. At the end of this operation, a sample ofN-acetylheparosan was withdrawn and purified according to the conditionsdescribed in "1--Production of an Escherichia coli (K5) SEBR 3282culture containing the enzyme--Step A--Determination of the distributionof molecular masses of N-acetylheparosan during the fermentation;b--Sample preparation".

The molecular masses of this sample were then assessed by ion exchangeHPLC.

Ion exchange HPLC operating conditions

HPLC for producing binary gradients (2 pumps)

Column: Pharmacia® Mono Q® HR 5/5, volume 1 ml

Detector: UV at 220 nm

Eluent: Ultrapurified water (A)

0.5M aqueous NaCl solution (B)

Use a gradient:

    ______________________________________                                               TIME (MIN)                                                                             % of B                                                        ______________________________________                                               0→2.00                                                                          30.00                                                                15.00    60.00                                                                17.00    100.00                                                               19.00    100.00                                                               20.00→25.00                                                                     30.00                                                         ______________________________________                                    

Flow rate: 1 ml/minute

calibration is performed using a series of oligosaccharides derived fromN-acetylheparosan, consisting of 6 to 14 disaccharide units.

The results obtained confirmed that the fragmentation of high molecularmass N-acetylheparosans into low molecular mass N-acetylheparosans isvirtually total (≧90%).

The predominant peaks of the fragments of which the low molecular massN-acetylheparosan is composed correspond to fragments of 8 to 12disaccharide units.

Hence the molecular mass of the low molecular mass N-acetylheparosanfragments is not modified by a further provision of eliminase. Theseresults confirm that the enzyme enables a virtually completefragmentation of a high molecular mass N-acetylheparosan to beperformed, and fragments having a molecular mass of approximately 5000Da or slightly less to be obtained.

EXAMPLE 2

Variation-controlled fragmentation of a high molecular massN-acetylheparosan--Production of an N-acetylheparosan of molecular massof approximately 7000 Da

Stage A

Mix 25 g of high molecular mass N-acetylheparosan, obtained according tothe PREPARATION, with an enzyme preparation solubilised with a detergentor alternatively by alkaline lysis (Example 1: 4--Solubilisation of theenzyme; 4.2 Solubilisation by alkaline lysis), obtained from 500 g offresh bacterial pellet which is itself obtained according to Example 1(1--Production of an Escherichia coli (KS) SEBR 3282culture containingthe enzyme. The pH is adjusted to 7 using 0.05M Tris-HCl buffer, pH 7.

Add an appropriate quantity of CaCl₂ so as to obtain a finalconcentration of 0.2M. Incubate at 37° C. for 24 hours.

Stage B

Centrifuge the mixture obtained for 5 minutes at 4000×g. Recover thesupernatant and filter it through a 0.2 μm membrane. Precipitate thefiltrate by adding 4 volumes of absolute ethanol, equivalent to a final80% of ethanol. Centrifuge at 4000×g for 5 minutes, remove thesupernatant and take the pellet up in ultrapurified water. Dialyse usinga Spectra-Por® sac (Spectrum) of cut-off threshold 10,000 Da overnightagainst ultrapurified water.

With magnetic stirring at 4° C., add concentrated sodium chloridesolution such that the final concentration is 0.5M, before precipitatingby adding 4 volumes of absolute ethanol per volume of salted dialysate.Centrifuge at 4000×g for 5 minutes and discard the supernatant.

Take the centrifugation pellets up in a buffer referred to as buffer D,of composition 20 mM Tris-HCl, pH 7.5, in the proportion of 100 ml/g.The solution obtained is chromatographed on a strong anion exchangecolumn containing an agarose matrix crosslinked with quaternary ammoniumgroups (Pharmacia® "Q Sepharose fast flow"), previously equilibratedwith buffer D in the proportion of 50 ml of gel per g of powder. Washthe gel with a sufficient quantity of buffer D for a return to thebase-line of UV detection at 214 nm, and then with a 25 mM solution ofpiperazine whose pH has been adjusted to 3.5. Elute with a solution ofpH 3.5 having the composition: 0.5M NaCl and 25 mM piperazine. Theeluate is neutralised using 5 M NaOH solution.

Precipitate by adding 4 volumes of ethanol. Centrifuge at 4000×g for 5minutes. Recover the centrifugation pellet and dry. An N-acetylheparosanconsisting of chains the majority of which have a molecular mass ofbetween 6000 and 8000 Da is thereby obtained.

EXAMPLE 3

Variation-controlled fragmentation of high molecular massN-acetylheparosan bound to a column of anion exchange resin in thepresence of the enzyme which is the subject of the present invention

A mass of Q Sepharose® resin (Pharmacia) is initially saturated withN-acetylheparosan predominantly of high molecular mass as described inthe PREPARATION. A membrane enzyme preparation, dissolved in a Tris-HClbuffer, pH 7.2 and containing 0.25M sodium chloride, is then introduced.The latter solution was prepared from a crude lysate as described inExample 1 (3--Localisation of the enzyme--Production of preparationscontaining this enzyme--Step B), but using a Tris-HCl buffer, pH 7.2containing 0.25M sodium chloride instead of ultrapurified water.

Reaction is allowed to take place for 60 hours. The N-acetylheparosanreleased as a result of the presence of 0.25M NaCl is isolated andanalysed. It is an N-acetylheparosan in which the majority of the chainshave a molecular weight of between 3200 and 4000 Da. On thechromatographic profile representing the distribution of molecularmasses, the maximum lies at 3600 Da.

The N-acetylheparosan bound to the resin is then eluted using 0.30Maqueous NaCl solution. A study of the chromatographic profilerepresenting the distribution of molecular masses enables it to beconfirmed that the maximum lies at 4300 Da. This product is anN-acetylheparosan consisting of chains longer than those of theN-acetylheparosan released.

According to another variant, it is possible to proceed as above butusing a membrane enzyme preparation dissolved in a Tris-HCl buffer, pH7.2 and containing 0.5M sodium chloride. In this case, a study of thechromatographic profile of the distribution of molecular masses of theN-acetylheparosan released enables it to be confirmed that the latter isan N-acetylheparosan consisting of longer chains, the maximum of thepeak lying at approximately 9200 Da. This demonstrates that, inconjunction with an anion exchange resin, it is possible to vary thefragmentation of N-acetylheparosan using membrane preparations having agiven NaCl concentration.

OTHER EXAMPLES OF USE OF THE ENZYME FOR THE FRAGMENTATION OF A HIGHMOLECULAR MASS N-ACETYLHEPAROSAN

The use of the enzyme which is the subject of the present invention inthe fragmentation of a high molecular mass N-acetylheparosan has alsobeen illustrated in Example 1, and in particular in:

* 2--In vivo demonstration of the enzyme in a fermenter.

* 3--Localisation of the enzyme--Production of preparations containingthis enzyme.

Step A--Production of a crude lysate possessing enzymatic activity.Assessment of its activity; b) demonstration of the fragmentationproduced by a crude lysate,

and

Step B--Production of a membrane preparation possessing enzymaticactivity--Assessment of its activity.

* 4--Solubilisation of the enzyme

The above represent processes for fragmentation of a high molecular massN-acetylheparosan which are also applicable at an industrial orsemi-industrial level.

We claim:
 1. An enzyme capable of fragmenting a high molecular massN-acetylheparosan, wherein said enzyme is isolated from Escherichia coli(K5) strain SEBR 3282, or from a spontaneous or induced mutant of saidstrain,has a molecular mass between 62,000 and 70,000 Da as determinedby gel permeation chromotography, has an isoelectric point in the pHrange between 4.7 and 5.4 pH units, and is an eliminase.
 2. An enzyme asclaimed in claim 1, wherein said enzyme:is of membrane origin, has atemperature of optimal functioning (maximal activity) in the region of37° C., and has an inactivated temperature at approximately 60° C., hasan optimal pH range for its optimal functioning between the values of pH6 and 7, has an optimal range of concentration of monovalent or divalentions for its optimal range of concentration of monovalent or divalentions for its optimal functioning in the vicinity of 0.2M.
 3. An enzymeas claimed in claim 1, wherein said enzyme:has a molecular mass between65,000 and 66,000 Da (±1500 Da), has an isoelectric point of 5.1 pHunits, has an optimal pH range for its optimal functioning between pH6.6 and pH 6.8, for its functioning, has an optimal range ofconcentration of monovalent ions at 0.25M, and an optimal range ofconcentration of divalent ions at 0.15M.
 4. A composition comprising acrude bacterial pellet obtained from a culture of Escherichia coli (K5)SEBR 3282 strain or a spontaneous or induced mutant of said strain,which pellet includes the enzyme of claim
 1. 5. A preparation comprisinga crude lysate obtained by lysis of the crude bacterial pellet of claim4, which crude lysate includes said enzyme.
 6. A preparation comprisinga refined lysate obtained by separation of cell membranes from the crudelysate of claim 5, which refined lysate includes said enzyme.
 7. Apreparation comprising the enzyme as claimed in claim 1 in solubilizedform.
 8. A preparation as claimed in claim 7, in which the enzyme hasbeen solubilized with anionic or cationic detergents.
 9. A preparationas claimed in claim 7, in which the enzyme has been solubilized bypartial lysis of a crude bacterial pellet, which includes said enzyme.10. A preparation comprising an enzyme as claimed in claim 1 incombination with another enzyme, an organic substance, or an inorganicsubstance.
 11. An enzyme as claimed in claim 1, obtained fromEscherichia coli (K5) strain Bi 8337-41 (010:K5:H4).
 12. An enzyme asclaimed in claim 1, which is an endo-β-eliminase.